The main function of analgesia is to alleviate pain or suppress its perception in a subject.
Pain can be defined in a variety of ways. One of them establishes pain as the perception by a subject of noxious stimuli which produces a withdrawal reaction by the subject. A variety of methods have long been available for the evaluation of subject's response to pain. In general, these experimental procedures must comply with several criteria in order to be of use. The effect to be measured must be unequivacally related to the pain experimented by the subject. The applied stimulus must produce a reproducible and measurable response, and ideally the response should be a single response. However, rarely do methods comply with the latter requirement.
It is recognized that there are at least two modes of pain known as pricking and burning pain. The sensation of pricking pain is one that reaches a sharp peak and subsides quickly while the one of burning pain has a slower onset, rises gradually, then plateaus but never reaches the same type of peak or intensity observed with pricking pain, and lasts for a longer period of time (Keith E. F., Amer. J. Pharm. 132:212-230 (1960)).
Pain has also been classified in accordance with other criteria (Pfeiffer, C. C. et al, Ann. N. Y. Acad. Sci. 51:21 (1948)) . "Supain" is used to describe superficial pain and is similar to the previously referred to as pricking pain. It can easily be elicited by pricking the skin with a needle. The second type of pain is "deepain" which is of an aching character. An example of this type is a tooth ache. Lastly, another type of pain is "sympain" which is exemplified by migraine headaches and is typically felt in the vessels of the temporal region. This pain is relieved by ganglionic blockade. The levels at which pain may be blocked are the receptors themselves. Many agents which block pain by acting superficially do so at the receptor itself. A second way of acting is that where the agents act at the internuncial pool of the spinal cord by blocking pain or raising the threshold for synaptic transmission. Centrally, the thalamus of the brain may also be involved in suppressing pain, or the cortex of the brain may be obtunded. Some analgesic agents may suppress muscle and joint pain. An example of these agents is salicylic acid which relieves pain from deep structures in muscular joint areas.
In order to study the mechanism of action of the different available drugs as well as novel drugs which are constantly being developed there is a broad background of techniques from which to select. However, all the available techniques have some beneficial and some detrimental characteristics. In general, the techniques can be divided into five groups:
1) Chemical methods PA1 2) Electrical methods PA1 3) Mechanical methods PA1 4) Thermal methods and PA1 5) Pharmacological methods
1) Chemical Methods
In general these methods rely on the application of a chemical compound to the skin of a subject. The simpler methods rely on the immersion of a part of the subject in a chemical which causes irritation to the skin. In another method the chemical irritant is administered subcutaneously. A more sophisticated method is one where a writhing agent is inserted in the peritoneal cavity of an animal and produces a characteristic writhing response which occurs five or more times in a period of 10 minutes immediately following the administration of the chemical (Siegmund et al, Proc. Soc. Exptl. Biol. Med. 95:729 (1957)). In general this type of method shows a lack of specificity in the response of the animals. It is unclear whether it is the irritation produced by the administered chemical or the release of some substance which is responsible for the observed effects.
2) Electrical Methods
The more primitive of these type of methods promote the painful stimulation of the skin to produce pain. In general the end point of this method is not sufficiently specific. A variation of this technique involves the application of an electrical current to the rat's scrotum by means of platinum electrodes. The current is increased until the animal squeals. Yet another electrical method is that where an electric current is applied to the pulp of a tooth (Koll, W. and Reffert, H., Arch. Exptl. Pathol. Pharmakol 190:687 (1938)). A modified version of this technique, and currently utilized, consists of delivering to an inlaid filling in a vital tooth a shock at various levels of intensity to determine the threshold of pain with and without a drug. This method has also been applied to humans. This method has the limitation imposed by the proximity of the filling to the pain receptors. In addition, different animals have different responses to the electrical stimulation of the pulp of the tooth and the responses are not always correlatable. The subject must be thus carefully watched and the electrodes require careful attachment for maintaining good electrical contact throughout the testing.
3) Mechanical Methods
The evaluation of the results of these methods is substantially subjective and does not permit the ready quantification thereof. These methods are therefore only applied in general to the primary screening of pain. Surgical blades adapted in various manners are utilized in the most primitive of these methods to produce a pain reaction. The blade is applied with a graded forceps and the amount of pressure necessary to elicit pain is expressed as the number of steps required in order to produce a squeak in the experimental animal . The pressure exercised by the blades of the forceps has also been measured by placing a dynamometer therebetween or between the handles of the forceps. Artery clamps or clips have also been utilized instead of forceps. In humans, mechanical pressure has been applied over bone structures or by eliciting visceral pain such as in the enteric canal or in the esophagus by introducing and inflating balloons therein. The amount of pressure required to produce pain can be measured by means of a manometer but the subject itself is the one to report the degree of pain. A more modern version of the method is that described by Randall-Selitto (Randall, L. O., and Selitto J. J., Arch. Int. Pharmacodyn CXI (4):409 (1957)). The pain threshold is measured in this method as the amount of pressure in mmHg required to induce a flight reaction (struggle) when applied to the foot of an experimental animal. Air pressure from an air line is admitted through a needle valve to a syringe into a pressure gauge which is connected by a T-tube. The syringe is mounted with a plunger downward, to which is connected a short bullet-shaped wooden peg. The pressure is applied through the wooden tip to the plantar surface of the rat's foot at a specified rate of mm Hg per second. The end point is said to have been reached when the rat starts struggling, as subjectively determined by an observer. The Randall-Selitto method has various drawbacks, including the fact that the mechanical stimuli activate both low and high threshold mechanoreceptive nerves in both cutaneous and non-cutaneous tissue, it requires that the experimental animal be restrained during the testing period, exhibiting therefore only limited behavioral movement and suffering from activation of stress-related physiologic responses such as sympatho-drenomedulary and pituitary-adrenal mediated stress responses, and the high degree of inaccuracy because of the subjective determination by an observer of the end point of the animal's response.
4) Thermal Methods
The simplest form of these methods is the hot plate technique originally described by Woolfe and McDonalds (Woolfe, G. and McDonalds, A. D. J. Pharmacol. Exptl. Therap. 80:300 (1944)). Originally, this method utilized a zinc plate with a lamp placed underneath. In a later modification it uses an electric lamp as the source of heat and a copper plate for the conduction of heat (Eddy, N. B. and Leimbach, D., J. Pharmacol EXPTL. Therap. 107:385 (1953)). The first sign of discomfort is usually expressed as an attempt to sit up and lick the forepaws by the experimental animal. This is taken to be an indication of a threshold under the predetermined conditions. Dancing and jumping about by an undrugged animal is taken as an indication of unbearable pain whereas drugged animals more commonly withdraw the hind paws and keep them close to their abdomen. Although widely used, this technique is also only recommended as a preliminary screening one because all points of contact between the animal and the hot plate are subjected to heat and the end-point is also determined subjectively by an observer. A hot wire technique consists of the application of heat from a wire coiled inside an asbestos plate. The animal's tail is placed in a channel made in the plate (Davis, O. L. et al, Brit. J. Pharmacol 1:255 (1946)). Yet another thermal method utilizing light from a headlamp focused on the tip of the tail of an animal (D'Amour, F. E., and Smith, D. L., J. Pharmacol. EXPTL. Therap. 72:74 (1941)). This method measures the time between the application of the heat and the flick of the tail and has therefore been called the "tail flick" method. With this method it is only possible to detect analgesic effects with large doses of chemical compounds such as aspirin and aminopyrine. However, these large doses only elicit a slight degree of analgesia. In general it is considered that this method is solely satisfactory for the detection of potent analgesics (e.g., opiates such as morphine) but unsatisfactory when applied to medium or weak analgesic agents. A further thermal method utilizes a light bulb focused on the loin of an animal which is protected by a plexiglass shield having a port and a shutter positioned between a lens and the animal. When the shutter is opened the timer starts and when the animal reacts the shutter is closed and the timer stopped (Ercoli, N., and Lewis, M. N., J. Pharmacol. EXPTL. Therap. 84:301 (1945)). This method applies an incident light the strength of which can be varied by means of a rheostat. This method is good for detecting potent analgesic substances but has the drawback that it only applies heat to one point of the body and the end-point is subjectively determined by an observer which operates the shutter and starts the timer. Weaker analgesic compounds such as aspirin show no effect when pain is measured by this method.
5) Pharmacological Methods
Typical representatives of this group are methods which rely on the administration of a substance to produce a rise in the temperature of an experimental animal. The skin temperature or the core temperature may be utilized. Yeast is a good example of a substance which can be used for producing an increase in temperature. The ability of a compound to inhibit this induced rise in temperature has been used as a means for evaluating antipyretic analgesic activity. In fact, the Randall-Selitto method described above (Randall, L. O. and Selitto, J. J., supra) is a combination of a mechanical method for evaluating analgesia and a physiological response method. It combines the administration of yeast as a phlogistic in the plantar surface of one paw of a rat and the use of a pressure device for eliciting a response. The threshold in the foot that has been injected with the yeast suspension drops to less than one half that of the control foot one hour after the injection of yeast.
An exhaustive comparison of the various method's known in the art was made by Taber (Taber, R. I., "Predictive Value of Analgesic Assays in Mice and Rats, in Narcotic Antagonists", edited by Braude et al, Advances in Biochemical Phytopharmacology, vol. 8, Raven Press, New York (1974), p. 191)). The scoring of most of the major techniques according to the methods rating scales are listed in the following table taken from Taber (Taber, R. I., supra) and are meant to provide a summary of the foregoing discussion.
TABLE 1 __________________________________________________________________________ Comparison of analgesic tests by rating scale Thermal Mechanical Chemical Electric Mouse Rat Mouse Rat Mouse Rat Mouse Criteria* hot-plate tail-flick tail-flick paw yeast writhing flick-jump tail-shock __________________________________________________________________________ Sensitivity a 2 4 2 4 4 4 3 b 2 2 1 3 4 3 3 c 2 1 2 1 0 2 1 d 1 2 0 2 2 2 2 Simplicity a 2 2 2 2 2 0 1 b 2 2 2 1 2 0 1 Reproducibility 4 4 4 4 4 1 2 Total Score 15 17 13 17 18 12 13 __________________________________________________________________________ *See Table 2.
The electrical methods, while promising, are of substantially no use because they lack simplicity and reproducibility. The remaining methods, however, score low in terms of sensitivity. General criteria for the scoring of the various methods are listed in Table 2 provided hereinbelow which has also been obtained from Taber (Taber, R. I., supra).
TABLE 2 ______________________________________ Analgesic method criteria rating scale Criteria Score ______________________________________ Sensitivity a. Is it sensitive to 1 for each class 4 (max. score) known analgesics? b. How sensitive is it to Median of drug 4 (max. score) doses in the clinical sensitivity range? No Questionable Yes c. Is it insensitive to 0 1 2 nonanalgesics? d. Is there an established, 0 1 2 clear-cut relationship between intensity and end point? Simplicity a. Does it require tech- 0 1 2 nical expertise? b. Does it require sophis- 0 1 2 ticated instrumenta- tion? Reproducibility How many labora- 0-4 4 tories have success- fully used the tech- nique ______________________________________
In general, the mouse writhing and the Randall-Selitto methods can detect antiinflammatory drugs. However, they do not provide for the automated detection of the end-point signal and are subject to substantial observer bias. On the other hand the rat tail flick method has an automated end-point but is very insensitive to various drugs such as antiinflammatory drugs, it lacks a within animal design and tests a particularly insensitive type of skin such as the one occurring in the tail of an animal. This type of skin is scaly and not very responsive to thermal stimulation.
Thus, despite the advantages of the various thermal stimulation methods (Dubner, R., et al, "Responses of Facial Cuntoneous Thermosensitive and Mechenosensive Afferent Fibers in the Monkey to Noxious Heat Stimulation", Adv. Neurol 4:61-71 (1974)), no good behavioral method exists for the quantitation of thermal nociception of hyperalgesia in a subject such as an animal model. The only method capable of quantitating behavioral responses to cutaneous hyperalgesia has been the Randall-Selitto method described above. This method has the various already indicated drawbacks. In addition, this method does not permit the simultaneous measurement of parameters other than the nociceptive threshold in the experimental animal.
Accordingly, there is still a need for a novel and simple method for the qualitative and/or quantitative in vivo measurement of responses to thermal stimulation in an unrestrained subject which overcomes the drawbacks of the prior art methods. Such method should ideally provide an automated detection of the subject's behavioral end-point, permit the use of unrestrained and unstressed animals, and optionally a within subject control, all of which and in the removal of observer bias and between animal variation while providing a powerful tool for distinguishing between centrally mediated and peripherally mediated responses to thermal stimulation.